1. Technical Field
The invention relates to the use of recombinant techniques to provide modified protein sequences resistant to oxidation, i.e., oxidation-receptive proteins. More specifically, the invention relates to providing proteins which retain useful biological activity, but which contain conservative amino acid substitutions for specific methionine residues that are particularly susceptible to oxidation.
2. Background Art
The fact that different proteins undergo varying degrees of modification during purification is well known, but not well understood. Certain proteins are known to be unusually susceptible to thermal denaturation or to proteolytic cleavage; other contain reactive amino acid side chains located in positions which render them particularly susceptible to chemical modification, including oxidation. In general, it is not possible to predict from the amino acid sequence the extent to which any of the above modifications will occur.
This problem is compounded because ordinary preparative methods for protein purification may lack the resolution to detect or separate small amounts of impurities. Such commonly employed techniques and criteria for homogeneity as electrophoresis, gel filtration, and ion exchange chromatography may fail to reveal the presence of small amounts of contaminating material which can be shown to be present by more sensitive analytical techniques, such as reverse phase high performance liquid chromatography (RP-HPLC).
Floor, E., et al., Anal. Biochem. (1980) 101:498-503 describes an illustration of this problem. An undecaprotein which has neurotransmitter properties, substance P, containing a carboxy terminal methionine that is oxidizable to methionine sulfoxide in dilute solutions exposed to air, elutes from ion exchange columns as a single peak, but reveals the presence of the sulfoxide-containing contaminant when subjected to RP-HPLC. Similarly, Frelinger, A. L., et al., J. Biol. Chem. (1984) 259:5507-5513 showed that bovine parathyroid hormone which appeared homogeneous by conventional criteria could be shown by RP-HPLC analysis to contain sequences with either one or both of the methionine residues oxidized to the sulfoxide. The proteins were eluted from RP-HPLC in an order directly proportional to the number of methionine residues oxidized.
Recently, Rosenberg, S., et al., Nature (1984) 312:77-80 produced a mutagenized human alpha-1 anti-trypsin in yeast which contained a valine residue in place of the methionine at the active site, and which retained significant biological activity. This substitution was made to prevent the observed decrease in inhibitory activity toward elastase, caused by oxidation of this methionine, which destroys the protective function of alpha-1 anti-trypsin in the lungs. The valine substitution resulted in an active anti-trypsin which was, unlike the native form, resistant to oxidation by chemical oxidants which may be similar to those released by leukocytes or present in cigarette smoke. Hence, by making this substitution, it is believed that the levels of anti-trypsin needed to be administered could be reduced. Carrell, R., Nature (1984) 312:77-80 discloses uses of .alpha.-antitrypsin mutants such as the Rosenberg et al. mutant in which methionine is replaced by valine. Transgene S.A. has replaced the methionine of .alpha.-antitrypsin with arginine, presented on May 20, 1985.
When a methionine is located at or near an active site, as in alpha-1 anti-trypsin, and is clearly crucial to biological activity, its oxidation to the sulfoxide might be expected to be detrimental. However, the effects of oxidation during purification of proteins containing several methionine residues which are not at the active site is less clear. Data for a number of proteins containing oxidized methionine residues show this oxidation to have varying degrees of negative effects on activity. At a minimum, the putatively pure protein obtained will, if the oxidized form is not separated away, contain a contaminant, and will therefore not be homogeneous. The effect of the presence of such contaminant(s) per se in, for example, pharmaceutical protein preparations, may be undesirable. Further, the presence of an oxidized methionine may cause other structural changes in the desired protein, such as decreased solubility, increased proteolytic lability, or the inhibition of formation of a required disulfide bond, which may, in turn, have additional side effects, such as the formation of dimeric and higher aggregated forms. Such impurities may be partly responsible for the often observed immunogenicity of parenterally administered proteins, or for other deleterious physiological reactions.
EP 0,130,756 published Jan. 9, 1985 discloses replacing methionine at position 222 of subtilisin with an amino acid such as alanine to provide resistance to oxidation. Estell, D. et al., J. Biol. Chem. (1985) 260:6518-6521 discloses mutant subtilisin enzymes whose methionine residues are susceptible to oxidation.
The present invention provides a method for protecting a therapeutic protein which normally contains methionines from oxidation to the methionine sulfoxide during fermentation, purification, storage, and clinical use, by substituting a conservative amino acid for any methionine particularly susceptible to oxidation. Thus protected, the muteins retain useful biological activity and exhibit enhanced chemical stability. They are not subject to contamination by either modified forms containing the methionine sulfoxide per se, or by side products whose formation is the result thereof.